Puerarin is extracted from puerarialobata (Willd.) Ohwi and P. thomaonii Benth, and is isoflavonoid C-glycosides with the chemical name 7,4′-dyhydroxy-8-β-D-glucosyl isoflavone, appearing as a white needle shaped crystal. It is dissolvable in water, but with a low solubility (6.24 g/L), and its water solution is colorless or slightly yellow. Its molecular weight is 416.37. Puerarin is one of the main active components in the traditional Chinese medicine Radix Puerariae, a natural low toxicity and effective medicine for treating cardio-cerebral vascular system diseases, with extensive pharmacological action. It has been clinically used to reduce blood pressure, slow down heart beat rate, and lower myocardial oxygen consumption. It can dilate coronary arteries and improve the metabolism of normal and ischemic myocardium. It has effect on cerebral circulation, peripheral vascular and micro-circulation. In addition, puerarin has the effects of controlling the blood sugar level, and anti-oxidation and anti-tumor (Yao Dan and Ding Xiansheng, Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13, 468-474.).
At present, puerarin formulations include liquid injection, eye drops and lyophilized powder injection, and in actual clinical application, it is mainly administered by injection (Wu Yanhong, Su Ziren, Lai Xiaoping, et la. Traditional Chinese Drug Research & Clinical Pharmacology, 2004, 15 (4):259-261.). As the solubility of puerarin in water is low, in clinical application, a cosolvent is required to raise the solubility. In the present time, a high concentration of propylene glycol is normally added as the cosolvent, thereby not only increasing the cost, but also making filtration more difficult in production due to a high viscosity. Furthermore, it reduces safety and performance of the medicine due to increased contents of insoluble impurity, causing some toxic side effect to human body. Thus, for making it more convenient in use, there is a need to improve the solubility and bioavailability of puerarin. According to relevant literatures, the main methods used today include glycosylation of puerarin or using a special formulation to effectively improve the puerarin solubility. Up to date, the reports on glycosylation of puerarin include the following: Li et al (Li D, Park S H, Shim J H, et al. Carbohyd Res, 2004, 339, 2789˜2797.) in 2004 first reported glycosylating puerarin using in vitro enzyme method, glucosyl-α-(1, 6)-puerarin (CASRN: 824959-75-3) and maltosyl-α-(1,6)-puerarin (CASRN: 824959-76-4) of glycosyl substitute on C-6″ hydroxyl group was obtained by catalytic composition of maltogenic amylase originated from bacillus thermophilus. The catalytic reaction took place with 1% (w/v) puerarin and 3% (w/v) soluble starch as the glycosyl donor, for 45 min at 55° C., and the product yield was 70%. Both converted products were separated and purified by a preparative HPLC method, and their water solubility was increased by respectively 14 times and 168 times as compared with puerarin. Jiang Jierong et al (Jiang J R, Yuan S, Ding J F, et al. Appl Microbiol Biotechnol, 2008, 81, 647-657.) used microbacterium oxydans to glycosylate puerarin, and obtained 7-O-glucoside puerarin (CASRN: 1163249-06-6) and 7-O-isomaltoside puerarin (CASRN: 1163249-07-7). The catalytic reaction took place with 4 g/L puerarin, 50 g/L cane sugar as glycosyl donor, at 30° C. for 48 h, the 7-O-glucoside puerarin mole conversion rate was 40%, and 7-O-isomaltoside puerarin mole conversion rate was 5%. Both converted products were separated and purified by a preparative HPLC method, and their water solubility was increased by respectively 18 times and 100 times as compared with puerarin. Huang et al (Huang W, Ochiai H, Zhang X Y, et al. Carbohyd Res, 2008, 343, 2903-2913.) used a acetyl galactosidase to convert puerarin by glycosylation in 20% DMSO with trimannose acetamido glucose oxazoline as glycosyl donor, and obtained the corresponding glycosidated compound Puerarin-GlcNAcMan3 (CASRN: 1093135-90-0). The catalytic reaction was conducted with 4.16 g/L puerarin and 13 g/L trimannose acetamido glucose oxazoline as a glycosyl donor, for 2 h at 23° C., Puerarin-GlcNAcMan3 mole conversion rate was 60%, but the preparation and relevant properties of this glycosylated product were not reported. Choi et al (Choi C H, Kim S H, Jang J H, et al. J Sci Food Agric, 2010, 90:1179-1184.) used malto-amylase (BSMA) glycosylated puerarin originating from fat bacillus thermophilus, and respectively obtained glucosyl-α-(1,6)-puerarin, maltosyl-α-(1,6)-puerarin and glucosyl-α-(1,3)-puerarin (CASRN: 1219937-73-1), with total yield rate of products of 56.7% at 10 g/L puerarin. Yu et al (Yu C G, Xu H D, Huang G D, et al. Appl Microbiol Biotechnol, 2010, 86:863-870.) treated microbacterium oxydans with 40% ethanol, changed the cellular permeability, and microbacterium oxydans can convert 7-O-glucoside puerarin to 7-O-fructoside puerarin (CASRN: 1223091-93-7). Zhang Lianwen et al (China Invention Patent Application No.: 200910068855.7; Publication No.: CN 101575631A) glycosylated puerarin with galactose transferase, in the catalytic reaction, with 9.12 g/L puerarin, 4 g/L UDP-galactose as glycosyl donor, semi-emulsified glycosyl-α-(1,4)-puerarin (CASRN: 1196677-60-7) was obtained, and its solubility is 12 times that of puerarin.
After puerarin is glycosylated, its water solubility is substantially increased. The molecular structure changes due to glycosylation do not affect the potency of puerarin, thus providing a puerarin glycosylated compound which can be administered at high concentrations. The relevant reports are as follows: Chung et al (Chung M J, Sung N J, Park C S, et al. Eur J Pharmacol, 2008, 578, 159-170.) used the mixture of equimolar glucosyl-α-(1,6)-puerarin (CASRN: 824959-75-3) and maltosyl-α-(1,6)-puerarin (CASRN: 824959-76-4) as water soluble puerarin glycosylation product, and the pharmacological experiment in HepG2 cells and C57 BL/6J mice showed that: the water soluble puerarin glycosylation product maintained the same potency as puerarin to resist oxidation activity and lower LDL oxidation. The in vitro pharmacokinetic experiment conducted by Jiang Jierong et al (Jiang J R, Yuan S, Ding J F, et al. Appl Microbiol Biotechnol, 2008, 81, 647-657.) with 7-O-glucoside puerarin (CASRN: 1163249-06-6) showed that: as compared with puerarin, 7-O-glucoside puerarin demonstrated better pharmacokinetic performance, the plasma half-life (t1/2) and mean retaining time (MRT) of 7-O-glucoside puerarin were respectively 2 times and 2.8 times that of puerarin, and this performance could possibly increase the bio-availability of 7-O-glucoside puerarin. Yuan Sheng et al (China Invention Patent Application No.: 200710021700.9) prepared drug to treat and prevent cardio-cerebral vascular diseases with 7-O-glucoside puerarin or 7-O-isomaltoside puerarin and their drug combination. Zhang Lianwen et al (China Invention Patent Application No.: 200910068855.7; Publication No.: CN 101575631A) compared the vasodilatation effect of puerarin and semi-emulsified glycosyl-α-(1,4)-puerarin on vascular smooth muscle with the aorta vascular smooth muscle as the physiological model, and found that semi-emulsified glycosyl-α-(1,4)-puerarin could produce better vasodilatation effect than puerarin.
Due to relatively poor water solubility of puerarin, however, the above mentioned bioconversion method of puerarin glycosylation has limitations because the puerarin concentration is generally low before conversion (all below 10 g/L), and the mole conversion rate of products is also low (all below 70%). These restrictions have made it quite difficult to obtain a sufficient amount of products for pharmacological experiment on related cardio-cerebral vascular and tumor diseases and the subsequent commercial production.